Identification of new amoebae strains in farmed rainbow trout (Oncorhynchus mykiss) affected by Nodular Gill Disease (NGD).

Introduction: The Nodular Gill Disease (NGD) is an emerging disease caused by amoebic infestations in farms of freshwater salmonids, resulting in huge economic losses. Nevertheless, unambiguous identification of the pathogenic amoebae has not yet been achieved. The aim was to identify the amoebae species involved in periodic NGD episodes in 2 rainbow trout (Oncorhynchus mykiss) farms in Northern Italy between February and April 2021. Methods: Four episodes were monitored and a total of 88 fish were euthanized and their gills evaluated by macroscopic, microscopic, and histological examination, with the assessment of 6-grade lesion scores. A portion of the second left gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoebae isolation, cultivation, and identification with SSU rRNA sequencing. Results: Histology confirmed moderate to severe NGD-related lesions and mild to moderate amoebae infestation. The presence of parasitic amoebae was significantly correlated with lesions severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed infection of Naegleria sp. strain GERK, two amoebae strains from Hartmannelidae, vannelid amoebae from the genus Ripella, and cercozoan amoeba Rosculus. Conclusions: The results highlight the utility of histopathology and SSU rDNA sequencing in NGD evaluation. The identification of known and new amoebae highlights the multi-etiological origin of this pathology, although the pathogenic role of each amoeba remains undefined.